CN107446897A - A kind of fish liver crude enzyme liquid freeze-dried powder and preparation method thereof - Google Patents
A kind of fish liver crude enzyme liquid freeze-dried powder and preparation method thereof Download PDFInfo
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- CN107446897A CN107446897A CN201710675790.7A CN201710675790A CN107446897A CN 107446897 A CN107446897 A CN 107446897A CN 201710675790 A CN201710675790 A CN 201710675790A CN 107446897 A CN107446897 A CN 107446897A
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Abstract
The invention discloses fish liver crude enzyme liquid freeze-dried powder and its preparation method and application.The fish liver crude enzyme liquid freeze-dried powder is the dry powder product of fish liver crude enzyme liquid.The present invention discloses the preparation method of the fish liver crude enzyme liquid freeze-dried powder, and the fish liver crude enzyme liquid of brand-new is prepared into fish liver crude enzyme liquid freeze-dried powder with long preservation period by freeze drier.The fish liver crude enzyme liquid freeze-dried powder of the present invention can apply to the measure of the Yi Fen azolactones ethoxyresorufin O-deethylase of 7 ethyoxyl 3, glutathione S-transferase, the detection of superoxide dismutase isoreactivity and reduced glutathione equal size.
Description
Technical field
The invention belongs to biochemical field, and in particular to a kind of fish liver crude enzyme liquid freeze-dried powder and preparation method thereof and should
With.
Background technology
Microsome be cell by homogenate it is broken when, the folliculus that oneself is reclosed after the membrane structure rupture of endomembrane system
Bubble (mainly endoplasmic reticulum), diameter about 100nm or so of these vesicles, is heterogeneous aggregate, referred to as micro-
Plastochondria.Microsome mainly contains cytochrome P 450 Enzyme (CYP) and antioxidant system albumen.Fish liver crude enzyme liquid is fish liver
The supernatant obtained after the broken centrifugation of homogenate, is mainly made up of microsome, containing a variety of biotransferases and albumen, such as 7- ethoxies
Base -3- Yi Fen azolactones ethoxyresorufin O-deethylases (7-ethoxyresorufin-o-deethylase, EROD), glutathione sulphur-transfer
Enzyme (glutathione S-transferase, GST), superoxide dismutase (superoxide dismutase, SOD) and
Glutathione (glutathione, GSH) etc..Research finds Cytochrome P450 (such as：EROD etc.) and antioxidant system enzyme system
(such as：GST, SOD etc.) and polyphenoils is (such as：GSH etc.) to pollutant (such as heavy metal, brominated flame-retardant, polycyclic in environment
Aromatic hydrocarbons, Polychlorinated biphenyls etc.) there is certain response relation.Existing many scholars by detect the EROD in fish liver crude enzyme liquid,
The change of the active and GSH of the enzymes such as GST, SOD etc. content carrys out indicative for environments situation, but fish liver crude enzyme liquid is liquid condition,
Be inconvenient to store with quantitative, and with the extension of holding time, easy inactivation, and enzyme source is used as using fish liver crude enzyme liquid freeze-dried powder
There is not been reported for the research of progress enzymatic activity and GAP-associated protein GAP assay.
The content of the invention
It is an object of the invention in place of overcome the deficiencies in the prior art, there is provided fish liver crude enzyme liquid freeze-dried powder and its system
Preparation Method and application.
One of the technical solution adopted for the present invention to solve the technical problems is：
Fish liver crude enzyme liquid freeze-dried powder is obtained dry powder product after the freeze-drying of fish liver crude enzyme liquid.
Wherein, the fish liver crude enzyme liquid freeze-dried powder is the dry powder system obtained after brand-new fish liver crude enzyme liquid is freeze-dried
Product.
The two of the technical solution adopted for the present invention to solve the technical problems are：
The preparation method of above-mentioned fish liver crude enzyme liquid freeze-dried powder, including：
1) cut open after fish takes out liver and (avoid staving fish courage), washed with 0.10~0.30% KCl solution of precooling, filter paper
Sop up bloodstain.Weigh, by 1:5 (W/V) ratio adds the PBS (pH 7.0~8.0) of precooling, is homogenized under ice bath.Homogenate is 0
At~8 DEG C, 8000~12000g centrifuges 10~30min, takes supernatant to dispense.
2) by the fish liver crude enzyme liquid being prepared in -80~-40 DEG C of refrigerators or liquid nitrogen cryopreservation 24~72 hours, to thick enzyme
Liquid fully charge.
3) the fish liver crude enzyme liquid freeze drier of freezing is dried 24~72 under -80~-40 DEG C and vacuum condition
Hour to fish liver crude enzyme liquid is changed into dry dry powder-shaped, in -20~-4 DEG C of preservations.
The three of the technical solution adopted for the present invention to solve the technical problems are：
Above-mentioned fish liver crude enzyme liquid freeze-dried powder is in 7- ethyoxyl -3- Yi Fen azolactones ethoxyresorufin O-deethylase, glutathione sulphur-transfer
Application in the measure of enzyme, the detection of superoxide dismutase isoreactivity and glutathione equal size.
Compared with background technology, it has the following advantages that the technical program：
1. the invention provides fish liver crude enzyme liquid freeze-dried powder and preparation method thereof.
2. of the invention dried brand-new fish liver crude enzyme liquid by freeze drier under -80~-40 DEG C and vacuum condition
It is changed into dry dry powder-shaped to fish liver crude enzyme liquid within 24~72 hours, for traditional fish liver crude enzyme liquid, its dry powder
Form is more convenient storage than the form of liquid, quantified, and stability is preferable, avoids the fish liver crude enzyme liquid of traditional freezen protective
Influence of the frozen-thaw process to enzymatic activity during use.
3. the fish liver crude enzyme liquid freeze-dried powder of the present invention can be used for 7- ethyoxyl -3- Yi Fen azolactones ethoxyresorufin O-deethylase, paddy
The measure of the sweet peptide sulfurtransferase of Guang, the detection of superoxide dismutase isoreactivity and glutathione equal size.
Brief description of the drawings
The invention will be further described with reference to the accompanying drawings and examples.
Fig. 1 is the fish liver crude enzyme liquid freeze-dried powder sample drawing obtained in embodiment.
Fig. 2 is EROD enzyme of the fish liver crude enzyme liquid with fish liver crude enzyme liquid freeze-dried powder of the different batches obtained in experimental example
Expression activitiy, wherein abscissa is fish liver enzyme source batch, and ordinate is the activity of EROD enzymes.
Embodiment
Present disclosure is illustrated below by embodiment.
Embodiment：Prepare fish liver crude enzyme liquid freeze-dried powder
Cut open after fish (Tilapia mossambica) takes out liver and (avoid staving fish courage), washed with 0.15% KCl solution of precooling, filter paper
Sop up bloodstain.Weigh, be 13.8g, shred, by 1:5 (W/V) ratio adds PBS (pH 7.4) 69.0mL of precooling, under ice bath
It is homogenized 3min.Homogenate 10000g centrifugation 20min, takes supernatant to dispense at 4 DEG C.The fish liver crude enzyme liquid that will be prepared
It is placed in liquid nitrogen cryopreservation 24 hours.The fish liver crude enzyme liquid freeze drier of freezing is dried 36 under -80 DEG C and vacuum condition
Hour, dried powder, as fish liver crude enzyme liquid freeze-dried powder are taken out, is placed in -20 DEG C of preservations.
The fish liver crude enzyme liquid freeze-dried powder sample drawing being prepared is as shown in Figure 1.
Above-described embodiment can realize the technique effect of following experimental examples：
Experimental example 1：The EROD enzyme assays of fish liver crude enzyme liquid freeze-dried powder
(1) preparation of samples：10mg fish liver crude enzyme liquid freeze-dried powders are taken, 50mg/mL is dissolved into 200 μ L pH 7.5 PBS
Crude enzyme liquid.
(2) active testing：At room temperature, crude enzyme liquid 20 μ L, 0.01mol/L PBS (pH 7.5) 160 are added in 96 orifice plates
μ L, 0.5mmol/L the μ L of 7- ethyoxyls -3- Yi Fen azolactones (ERF) solution 5, it is eventually adding 0.5mmol/L NADPH solution
40 μ L, is mixed immediately using 532nm as excitation wavelength, using 580nm as launch wavelength, determine fluorescence intensity.Determined once per 30s
Fluorescent value, METHOD FOR CONTINUOUS DETERMINATION 25min.
7- ethyoxyl -3- Yi Fen azolactone ethoxyresorufin O-deethylase the activity measured is：0.59pmol/min/mg pro
Experimental example 2：The EROD activity ratios of different batches fish liver crude enzyme liquid and fish liver crude enzyme liquid freeze-dried powder compared with
(1) the EROD enzyme assays in different batches fish liver crude enzyme liquid：Operation is with reference to the step of experimental example 1 (2).
(2) the EROD enzyme assays in different batches fish liver crude enzyme liquid freeze-dried powder：Operation is with reference to the step of experimental example 1
(1)、(2)。
The experimental result of the EROD enzyme assays of different batches fish liver crude enzyme liquid and fish liver crude enzyme liquid freeze-dried powder is such as
Shown in Fig. 2.
Above-mentioned experimental result is shown：The EROD enzymatic activitys of fish liver crude enzyme liquid freeze-dried powder are unlike thick enzyme made from each batch
Liquid it is active low, substantially freeze-dried powder activity for crude enzyme liquid activity 77-120%, illustrate the freeze-dried powder storage new as crude enzyme liquid
The form of depositing has feasibility.
Experimental example 3：The GST enzyme assays of fish liver crude enzyme liquid freeze-dried powder
10mg fish liver crude enzyme liquid freeze-dried powders are taken, 50mg/mL crude enzyme liquid is dissolved into 200 μ L pH 6.5 PBS.Room
Under temperature, crude enzyme liquid 10 μ L, 0.01mol/L PBS (pH 6.5) 170 μ L, 30mmol/L GSH solution 10 are added in 96 orifice plates
μ L, the 30mmol/L μ L of CDNB solution 10 are eventually adding, mixed.Non- enzymatic reaction is not added with enzyme liquid, immediately per 30s under 340nm
Determine a light absorption value, METHOD FOR CONTINUOUS DETERMINATION 5min.
The GST enzymatic activitys measured are：15U/mL
Experimental example 4：The SOD enzyme activity measure of fish liver crude enzyme liquid freeze-dried powder
10mg fish liver crude enzyme liquid freeze-dried powders are taken, 50mg/mL crude enzyme liquid is dissolved into 200 μ L pH 8.2 PBS.Room
Under temperature, the 0.01mol/L μ L of Tris-HCl solution (pH 8.2) 100 are added in 96 orifice plates, the μ L of ultra-pure water 90, are eventually adding
The 4.5mmol/L μ L of pyrogallol solution 10, mix and determine a light absorption value per 30s at 325 nm immediately, determine 5min.Meter
Mouse thymus cells speed is calculated, the μ L of supernatant 10 is added by above step, autoxidation speed is suppressed about 50%.
The SOD enzyme activity measured is：110U/mL
Experimental example 5：The GSH assays of fish liver crude enzyme liquid freeze-dried powder
10mg fish liver crude enzyme liquid freeze-dried powders are taken, 50mg/mL crude enzyme liquid is dissolved into 200 μ L pH 8.0 PBS.Room
Under temperature, 10 μ L crude enzyme liquids, 180 μ L 0.01mol/L phosphate buffer solution (pH 8.0) and 10 μ L neighbour's benzene are added in 96 orifice plates
The ethanol solution (1mg/mL) of dicarbaldehyde, fully mix, 20min. is placed at room temperature using 340nm as excitation wavelength, under 430nm
Determine fluorescence intensity.
The GSH measured content is：0.8mmol/L
It is described above, preferred embodiments only of the present invention, therefore the scope that the present invention is implemented can not be limited according to this, i.e., according to this
The equivalent changes and modifications that patent of invention scope and description are made, it should all still fall within the range of the present invention covers.
Claims (4)
1. fish liver crude enzyme liquid freeze-dried powder, it is characterised in that：The liver crude enzyme liquid freeze-dried powder is that the freezing of fish liver crude enzyme liquid is dry
Dry powder product after dry.
2. fish liver crude enzyme liquid freeze-dried powder according to claim 1, it is characterised in that：The fish liver crude enzyme liquid freeze-dried powder
The dry powder product obtained after being freeze-dried for brand-new fish liver crude enzyme liquid.
3. the preparation method of fish liver crude enzyme liquid freeze-dried powder according to claim 1, it is characterised in that：
1) after cuing open fish taking-up liver, washed with 0.10~0.30% KCl solution of precooling, filter paper sops up bloodstain；Weigh, by 1:
5 (W/V) ratio adds the PBS (pH 7.0~8.0) of precooling, is homogenized under ice bath；Homogenate at 0~8 DEG C, 8000~
12000g centrifuges 10~30min, takes supernatant to dispense；
2) it is complete to crude enzyme liquid by the fish liver crude enzyme liquid being prepared in -80~-40 DEG C of refrigerators or liquid nitrogen cryopreservation 24~72 hours
Freeze entirely；
3) the fish liver crude enzyme liquid freeze drier of freezing is dried 24~72 hours under -80~-40 DEG C and vacuum condition
It is changed into dry dry powder-shaped to fish liver crude enzyme liquid, in -20~-4 DEG C of preservations.
4. fish liver crude enzyme liquid enzyme dry powder according to claim 1 is taking off ethyl including 7- ethyoxyl -3- Yi Fen azolactones
The detection of enzymatic activity including enzyme, glutathione S-transferase, superoxide dismutase and reduced glutathione content
Purposes in measure.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710675790.7A CN107446897A (en) | 2017-08-09 | 2017-08-09 | A kind of fish liver crude enzyme liquid freeze-dried powder and preparation method thereof |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002062969A9 (en) * | 2001-02-06 | 2002-10-17 | Massachusetts Inst Technology | Cellular reprogramming in peptide hydrogel and uses thereof |
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2017
- 2017-08-09 CN CN201710675790.7A patent/CN107446897A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002062969A9 (en) * | 2001-02-06 | 2002-10-17 | Massachusetts Inst Technology | Cellular reprogramming in peptide hydrogel and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| 曾青兰 等: "《生物制药工艺》", 29 February 2012, 华中科技大学出版社 * |
| 朱智 等: "EROD、GST测试法在评价污染物生态效应的研究", 《中国科技论文在线》 * |
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Application publication date: 20171208 |